Gelatin / antibacterial peptide covalent conjugate with antibacterial activity as well as preparation and application of gelatin / antibacterial peptide covalent conjugate

Abstract

The invention provides a gelatin / antibacterial peptide covalent conjugate with antibacterial activity as well as preparation and application of the gelatin / antibacterial peptide covalent conjugate. The preparation method comprises the following steps: adding a reaction medium into gelatin, and heating to fully dissolve the gelatin to obtain a gelatin solution; the preparation method comprises the following steps: adding antibacterial peptide into a gelatin solution, adding or not adding a functional reagent, fully dissolving, and adjusting the pH value to 3-6.5 or 9-12 to obtain a premixed solution; and adding a coupling agent into the premixed solution, carrying out a coupling reaction, after the reaction is finished, adjusting to be neutral, dialyzing, and freeze-drying to obtain the product. The preparation method has the characteristics of low cost, high efficiency and mild reaction, activation of amino groups or carboxyl groups of the gelatin and the antibacterial peptide can be realized by simply adjusting the acid-base property of a reaction system, and then covalent coupling of the gelatin, the antibacterial peptide and a coupling agent is realized, so that antibacterial peptide molecules are coupled to a gelatin molecular chain, and the antibacterial peptide is prepared. The gelatin is endowed with excellent antibacterial performance.

Description

Technical Field

[0001] The invention belongs to the technical field of macromolecular antibacterial biomaterials, and specifically comprises a gelatin / antibacterial peptide covalent coupling material with antibacterial activity, and its preparation and application. Background Art

[0002] Gelatin is a partial hydrolysis product of collagen after acid, alkali or enzyme treatment. It has good biocompatibility, biodegradability and low immunogenicity. It is a widely used biopolymer material and has been widely used in production and life fields such as photosensitive imaging, packaging materials, food, cosmetics, feed, etc. In the high value-added biomedical field, gelatin is also widely used in therapeutic drugs and equipment in dermatology, ophthalmology, orthopedics and other departments.

[0003] In the real production and life, microbial infection is a major problem that has troubled mankind for thousands of years. Food storage and circulation are contaminated by microorganisms, which leads to food corruption and deterioration. Humans may suffer from foodborne diseases if they eat contaminated food by mistake. At the same time, harmful microbial infection leads to a variety of human diseases. Surgical infection delays the healing of wounds and can even endanger life in severe cases. Gelatin does not have antibacterial activity. At the same time, because gelatin is composed of amino acids, it can also be used as a nutrient for microorganisms, which greatly limits the application of gelatin. In order to give gelatin antibacterial activity, people add antibacterial agents to gelatin products, including organic antibacterial materials, inorganic antibacterial materials, and natural antibacterial materials.

[0004] Antimicrobial peptides are a class of polypeptide substances with antimicrobial activity. They are widely present in bacteria, plants, and animals, and are important effector molecules of host cell innate immunity. Antimicrobial peptides have broad-spectrum antimicrobial properties, have good inhibitory effects on multi-drug resistant bacteria, and are not prone to drug resistance. They are expected to become a substitute for antibiotics and have received widespread attention from scientists. Natural preservatives such as ε-polylysine, natamycin, and nisin are antimicrobial peptides produced by Streptomyces and Streptococcus lactis, and have good inhibitory effects on a variety of bacteria and fungi.

[0005] At present, there are two main ways to prepare gelatin / antimicrobial peptide composite materials: 1) The first way is to physically mix gelatin and antimicrobial peptides; 2) The second way is to form a covalent conjugate with gelatin and antimicrobial peptides. Since there is no strong chemical bond between gelatin and antimicrobial peptides in the physical mixing method, the antimicrobial peptides are easily lost, causing the composite material to lose its antimicrobial activity. At the same time, antimicrobial peptides also have disadvantages such as easy degradation by enzymes, unstable activity, low bioavailability, certain hemolysis and cytotoxicity. For example, acidic polysaccharides, hydrochlorides, phosphates, copper ions, etc. will reduce the antimicrobial activity of ε-polylysine. At the same time, when ε-polylysine decomposes and the molecular weight is lower than 1300, ε-polylysine loses its antibacterial activity. The formation of a covalent conjugate between gelatin and antimicrobial peptides can overcome the disadvantages of the above-mentioned physical mixing and help stabilize the activity of antimicrobial peptides. The method reported in the literature currently uses gelatin and antimicrobial peptides as raw materials, activates the carboxyl group of gelatin or antimicrobial peptides through the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS) system, and reacts with the amino group of another reactant to form an amide bond to generate a covalent conjugate. However, the EDC / NHS reagent used in this method is expensive and is used in large quantities when activating the carboxyl group, thereby increasing the cost of antimicrobial gelatin. At the same time, this method is only suitable for generating conjugates under conditions of low reactant concentrations. When the gelatin concentration is increased, the gelatin quickly cross-links to generate a gel product.

[0006] Based on this, there is an urgent need to develop a method that is low in cost, simple to operate, highly efficient, and can be used for large-scale preparation of gelatin / antimicrobial peptide covalent conjugates. Summary of the invention

[0007] In view of the above problems existing in the prior art, the first object of the present invention is to provide a method for preparing a gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity. The preparation method has the characteristics of low cost, high efficiency and mild conditions. The gelatin / antimicrobial peptide covalent conjugate with antimicrobial properties is formed by activating the amino groups of gelatin and antimicrobial peptide under alkaline conditions, or activating the carboxyl groups of gelatin and antimicrobial peptide under acidic conditions, and then coupling with the epoxy groups at both ends of the coupling agent molecule; at the same time, the preparation method can realize the production of several grams to several hundred grams of gelatin raw materials, and can prepare gelatin / antimicrobial peptide covalent conjugates with antimicrobial activity on a large scale.

[0008] The second object of the present invention is to provide a gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity prepared by the above-mentioned preparation method. The gelatin / antimicrobial peptide covalent conjugate has high antimicrobial activity, and can also be endowed with new properties by introducing functional reagents, which is conducive to broadening the application range of the gelatin / antimicrobial peptide covalent conjugate.

[0009] The third object of the present invention is to provide a use of the gelatin / antimicrobial peptide covalent conjugate as described above in the preparation of antibacterial and / or antibacterial products.

[0010] To achieve the above first object, the technical solution adopted by the present invention includes:

[0011] The present invention discloses a method for preparing a gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity, comprising the following steps:

[0012] 1) adding a reaction medium to gelatin and heating the gelatin to fully dissolve the gelatin to obtain a gelatin solution;

[0013] 2) adding antimicrobial peptides to the gelatin solution, and then adding or not adding functional reagents, adjusting the pH to 3-6.5 or 9-12 after sufficient dissolution to obtain a premixed solution;

[0014] 3) Add a coupling agent to the premixed solution to carry out coupling reaction. After the reaction is completed, adjust to neutral, dialyze, and freeze-dry to obtain the product.

[0015] The invention realizes activation of the amino group or carboxyl group of gelatin and antimicrobial peptide by simply adjusting the acidity and alkalinity of the reaction system, and the activated gelatin and antimicrobial peptide are coupled through the epoxy groups at both ends of the coupling agent molecule, so that the antimicrobial peptide molecule is coupled to the gelatin molecular chain to form a gelatin / antimicrobial peptide covalent coupling compound with antimicrobial properties. From the perspective of cost, the preparation method abandons the EDC / NHS activation system used in the traditional technology, has lower cost and higher efficiency, can simply and quickly prepare the antimicrobial gelatin composite material at a higher gelatin concentration, and can avoid rapid cross-linking of gelatin to form a gel by coordinated control of the concentration of the gelatin solution and the ratio of gelatin / polylysine / coupling agent, so as to achieve the effect of maintaining the solution state in the whole preparation process.

[0016] Furthermore, the gelatin used in the present invention can be selected from gelatin commonly used in the art, and the source of the gelatin is not limited, including but not limited to one or more of gelatin from pigs, cattle, fish and poultry.

[0017] Furthermore, the mass fraction of gelatin in the gelatin solution is 2-20%, preferably 5-15%.

[0018] Furthermore, the reaction medium includes, but is not limited to, one or more of distilled water, sodium chloride aqueous solution, carbonate buffer, Tris-HCl solution, 2-morpholineethanesulfonic acid solution and acetate buffer.

[0019] Furthermore, when dissolving gelatin in step 1, the dissolving temperature is controlled to be 35-60°C, preferably 40-55°C.

[0020] Furthermore, the antimicrobial peptide used in the present invention can be a natural antimicrobial peptide or an artificially synthesized antimicrobial peptide, as long as it has a free amino group and / or carboxyl group. The antimicrobial peptide includes but is not limited to one or more of ε-polylysine or its salt, nisin or its salt, natamycin or its salt.

[0021] Furthermore, the mass ratio of the antimicrobial peptide to gelatin is 0.05-2:1, preferably 0.1-1:1.

[0022] Further, the functional agent includes but is not limited to one or more of an antioxidant enhancing agent, an antibacterial enhancing agent, a mechanical strength enhancing agent, and an extensibility enhancing agent; illustratively, the functional agent includes but is not limited to one or more of water-soluble chitosan, tea polyphenols, citric acid, tannic acid, hyaluronic acid, etc. To help these functional agents dissolve, solvents such as ethanol, ethylene glycol, and glycerol can be added in appropriate amounts. These solvents can be completely removed during the dialysis and drying steps and have no effect on biological safety.

[0023] Furthermore, the functional agent is 0-20wt% of the gelatin mass.

[0024] Furthermore, considering that gelatin is easily decomposed under excessively acidic or alkaline conditions, and the effect of acidity and alkalinity on the reaction vessel in future large-scale production, a more suitable pH range for step 2 is 4-6 or 10-11.

[0025] Further, the coupling agent has multiple epoxy groups, including but not limited to ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, pentaerythritol glycidyl ether, propylene glycol triglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, trimethylolpropane triglycidyl ether, four-arm polyethylene glycol glycidyl ether, etc., preferably one or more of ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, propylene glycol triglycidyl ether, and 1,4-butanediol diglycidyl ether with greater solubility in water.

[0026] Furthermore, the mass ratio of the coupling agent to gelatin is 0.01-0.10:1, preferably 0.02-0.05:1.

[0027] Furthermore, the reaction temperature of the coupling reaction is 35-60°C, preferably 40-55°C, and the reaction time is 8-48h, preferably 12-24h.

[0028] Furthermore, the dialysis process is to remove unreacted antimicrobial peptides and coupling agents to reduce their impact on subsequent applications, which can be controlled by the molecular weight cutoff of the dialysis bag. The temperature controlled during the dialysis process is room temperature-50°C, preferably 35-45°C, and the dialysis time is 1-5 days; the molecular weight cutoff of the dialysis bag is 20-40kDa.

[0029] To achieve the above second purpose, the technical solution adopted by the present invention includes:

[0030] The invention discloses a gelatin / antimicrobial peptide covalent coupling material with antimicrobial activity prepared by the above-mentioned preparation method.

[0031] To achieve the third objective above, the technical solution adopted by the present invention includes:

[0032] The present invention discloses an application of the gelatin / antimicrobial peptide covalent conjugate described above in the preparation of antibacterial and / or antibacterial products.

[0033] Beneficial effects of the present invention:

[0034] The invention discloses a method for preparing a gelatin / antimicrobial peptide covalent conjugate. The preparation method has the characteristics of low cost, high efficiency and mild reaction. The gelatin / antimicrobial peptide covalent conjugate with antimicrobial properties is formed by activating the amino groups of gelatin and the antimicrobial peptide under alkaline conditions or activating the carboxyl groups of gelatin and the antimicrobial peptide under acidic conditions, and then coupling with epoxy groups at both ends of a coupling agent molecule. At the same time, the preparation method can realize the production of several grams to several hundred grams of gelatin raw materials, and can prepare gelatin / antimicrobial peptide covalent conjugates with antimicrobial activity on a large scale.

[0035] The present invention can activate the amino or carboxyl groups of gelatin and antimicrobial peptides by simply adjusting the acidity and alkalinity of the reaction system. The activated gelatin and antimicrobial peptides are then coupled via the epoxy groups at both ends of the coupling agent molecules, thereby coupling the antimicrobial peptide molecules to the gelatin molecular chains to form gelatin / antimicrobial peptide covalent conjugates with antimicrobial properties. From a cost perspective, the preparation method abandons the EDC / NHS activation system used in traditional technologies, has lower costs and higher efficiency, and can simply and quickly prepare antimicrobial gelatin composite materials at a higher gelatin concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0037] Figure 1 The antibacterial photos of the gelatin / ε-polylysine covalent conjugates obtained in Examples 1-4 against Staphylococcus aureus and Escherichia coli are shown.

[0038] Figure 2 The infrared spectra of the gelatin / ε-polylysine covalent conjugate obtained in Example 1 are compared with those of gelatin and ε-polylysine.

[0039] Figure 3 The antibacterial photos of the gelatin / ε-polylysine covalent conjugate obtained in Example 5 against Staphylococcus aureus and Escherichia coli are shown.

[0040] Figure 4 The photographs showing the antibacterial effect of the gelatin / nisin Z covalent conjugate obtained in Example 6 on Staphylococcus aureus and Escherichia coli are shown. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be clear that the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] All numerical specifications herein (e.g., temperature, time, concentration, and weight, etc., including ranges for each thereof) are generally approximate values ​​that may be appropriately varied (+) or (-) in increments of 0.1 or 1.0. All numerical specifications may be understood as being preceded by the term "about."

[0043] Example 1

[0044] Dissolve 10g gelatin in 100mL 2% NaCl aqueous solution and dissolve at 40℃ for half an hour to form a gelatin solution; add 10g ε-polylysine hydrochloride to the obtained gelatin solution, continue stirring until dissolved, adjust the pH of the reaction system to pH = 10, and obtain a premixed solution. Add 0.2g ethylene glycol diglycidyl ether to the premixed solution, continue stirring and react at 40℃ for 24 hours. After the reaction is completed, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 5 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain.

[0045] Example 2

[0046] Dissolve 10g gelatin in 100mL 2% NaCl aqueous solution and dissolve at 40℃ for half an hour to form a gelatin solution; add 5g ε-polylysine hydrochloride to the obtained gelatin solution, continue stirring until dissolved, adjust the pH of the reaction system to pH = 10, and obtain a premixed solution. Add 0.2g ethylene glycol diglycidyl ether to the premixed solution, and continue stirring at 40℃ for 24 hours. After the reaction, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 3 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain.

[0047] Example 3

[0048] Dissolve 10g gelatin in 100mL 2% NaCl aqueous solution and dissolve at 40℃ for half an hour to form a gelatin solution; add 2.5g ε-polylysine hydrochloride to the obtained gelatin solution, continue stirring until dissolved, adjust the pH of the reaction system to pH = 10, and obtain a premixed solution. Add 0.2g ethylene glycol diglycidyl ether to the premixed solution, continue stirring at 40℃ for 24 hours. After the reaction is completed, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 3 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain.

[0049] Example 4

[0050] Dissolve 10g gelatin in 100mL 2% NaCl aqueous solution and dissolve at 40℃ for half an hour to form a gelatin solution; add 1.25g ε-polylysine hydrochloride to the obtained gelatin solution, continue stirring until dissolved, adjust the pH of the reaction system to pH = 10, and obtain a premixed solution. Add 0.2g ethylene glycol diglycidyl ether to the premixed solution, continue stirring and react at 40℃ for 24 hours. After the reaction is completed, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 3 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain.

[0051] Test Case

[0052] The KB paper disc method was used to investigate the antibacterial effects of gelatin / ε-polylysine covalent conjugates obtained with different raw material ratios on Staphylococcus aureus and Escherichia coli, with PBS solution as the control. Figure 1 The gelatin / ε-polylysine covalent conjugate showed obvious inhibition zones for Escherichia coli and Staphylococcus aureus. When the feed ratio of gelatin to ε-polylysine was changed, the prepared gelatin / ε-polylysine covalent conjugate had different minimum inhibitory concentrations and minimum bactericidal concentrations for Staphylococcus aureus and Escherichia coli. With the increase of ε-polylysine dosage, its minimum inhibitory concentration and minimum bactericidal concentration decreased, and a lower antibacterial gelatin concentration could inhibit and kill Escherichia coli and Staphylococcus aureus.

[0053] Table 1

[0054]

[0055] Figure 2 This is the Fourier transform infrared spectrum of the sample prepared in Example 1. As shown in the figure, gelatin and ε-polylysine undergo coupling reaction, with the peak at 3767 cm -1The NH infrared absorption peak at 1667 cm-1 shifts toward lower wavenumbers, indicating that the NH in the gelatin molecule undergoes a covalent reaction. -1 、1568cm -1 The absorption peak at 1541 cm-1 shifted toward the lower wavenumber direction, indicating that the amide bond in the gelatin molecule reacted. -1 The absorption peak at the wave number was significantly enhanced, indicating that the primary amino group in the gelatin molecule was coupled with the epoxy group and the primary amino group was converted into a secondary amide group.

[0056] Example 5

[0057] The preparation process is the same as that of Example 4, except that the preparation system is enlarged to 500 g of gelatin and 62.5 g of ε-polylysine hydrochloride, and the coupling agent is 10 g of ethylene glycol diglycidyl ether. The KB paper disc method shows that the gelatin / ε-polylysine covalent conjugate exhibits obvious inhibition zones against Escherichia coli and Staphylococcus aureus ( Figure 3 ). This indicates that the preparation method provided by the present invention can successfully prepare gelatin materials with antibacterial activity at a higher concentration and on a larger scale.

[0058] Example 6

[0059] Dissolve 10g gelatin in 200mL 2-morpholineethanesulfonic acid (MES) buffer and dissolve at 40℃ for half an hour to form a gelatin solution; add 100mL of an acidic solution containing 1.25g nisin (Nisin-Z) to the obtained gelatin solution, continue stirring until dissolved, adjust the pH of the reaction system to pH=5.5, and obtain a premixed solution. Add 0.2g polyethylene glycol diglycidyl ether (molecular weight 2000) to the premixed solution, and continue stirring at 50℃ for 24 hours. After the reaction is completed, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 3 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain. The KB paper disc method showed that the gelatin / nisin covalent conjugate showed a significant inhibition zone against Staphylococcus aureus, but had no significant antibacterial effect against Escherichia coli ( Figure 4 ). This is because nisin itself has an antibacterial effect only on Staphylococcus aureus, but not on Escherichia coli, and its properties are retained by forming a covalent conjugate with gelatin.

[0060] Example 7

[0061] Dissolve 10g gelatin in 100mL MES buffer and dissolve at 40℃ for half an hour to form a gelatin solution; add 2g ε-polylysine hydrochloride to the obtained gelatin solution, then add 100mL 2% water-soluble chitosan MES solution, continue stirring until dissolved, adjust the pH of the reaction system to pH = 5.5, and obtain a premixed solution. Add 0.3g 1,4-butanediol diglycidyl ether to the premixed solution, and continue stirring at 50℃ for 24 hours. After the reaction, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30kDa, dialyze at 40℃ for 5 days to remove unreacted polylysine and coupling agent, and then freeze at -80℃ and vacuum dry to obtain.

[0062] Comparative Example 1

[0063] 10g gelatin was dissolved in 100mL 2-morpholineethanesulfonic acid buffer at 40℃ for half an hour to form a gelatin solution. 0.25g ε-polylysine hydrochloride was added and stirred continuously to dissolve. The pH of the reaction system was adjusted to pH=5.5. 1g-10g EDC and 1g-10g NHS were added for coupling. The system formed a gel within a few minutes, and it was impossible to remove impurities and obtain powdered antibacterial gelatin material.

[0064] Comparative Example 2

[0065] Dissolve 2g gelatin in 100mL 2-morpholineethanesulfonic acid buffer and dissolve at 40℃ for half an hour to form a gelatin solution. Add 0.05g ε-polylysine hydrochloride, stir and dissolve continuously, adjust the pH of the reaction system to pH=5.5, add 0.2g-2g EDC and 0.2g-2g NHS for coupling, and stir and react at 40℃ for 24 hours. After the reaction, adjust the pH of the solution to neutral, dialyze with a dialysis bag with a molecular weight cutoff of 30,000 to remove unreacted polylysine, then freeze at -80℃ and vacuum dry to obtain. During the preparation process, the reaction system remains in a solution state and no gel is formed. This shows that when EDC / NHS is used for coupling, the low-concentration reaction system can always remain in a solution state.

[0066] Comparative Example 3

[0067] The preparation process is the same as that in Example 1, the only difference is that ε-polylysine hydrochloride is not added or 0.1 g ε-polylysine hydrochloride is added to participate in the reaction, and other conditions remain unchanged. After the coupling reaction, a large amount of gel-like substance is formed in the reaction system, which affects the next step of impurity removal and obtaining the powdered antibacterial gelatin material.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity, characterized in that: The steps include: 1) adding a reaction medium to gelatin and heating the gelatin to fully dissolve the gelatin to obtain a gelatin solution; 2) adding antimicrobial peptides to the gelatin solution, and then adding or not adding functional reagents, adjusting the pH to 3-6.5 or 9-12 after sufficient dissolution to obtain a premixed solution; 3) Add a coupling agent to the premixed solution to carry out coupling reaction. After the reaction is completed, adjust to neutral, dialyze, and freeze-dry to obtain the product.

2. The preparation method according to claim 1, characterized in that: The gelatin includes one or more of gelatin from pig, cattle, fish and poultry sources; Preferably, the mass fraction of gelatin in the gelatin solution is 2-20%, preferably 5-15%; Preferably, the reaction medium comprises one or more of distilled water, sodium chloride aqueous solution, carbonate buffer, Tris-HCl solution, 2-morpholineethanesulfonic acid solution and acetate buffer.

3. The preparation method according to claim 1, characterized in that: The antimicrobial peptides include one or more of ε-polylysine or its salt, nisin or its salt, natamycin or its salt; Preferably, the mass ratio of the antimicrobial peptide to gelatin is 0.05-2:1, preferably 0.1-1:

1.

4. The preparation method according to claim 1, characterized in that: The functional agent includes one or more of an antioxidant enhancing agent, an antibacterial enhancing agent, a mechanical strength enhancing agent, and an extensibility enhancing agent; Preferably, the functional agent is 0-20 wt% of the gelatin mass.

5. The preparation method according to claim 1, characterized in that: The pH adjusted in step 2 is 4-6 or 10-11.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the coupling agent to gelatin is 0.01-0.10:1, preferably 0.02-0.05:

1.

7. The preparation method according to claim 1, characterized in that: The reaction temperature of the coupling reaction is 35-60° C., and the reaction time is 8-48 h.

8. The preparation method according to claim 1, characterized in that: The dialysis temperature is room temperature-50°C, preferably 35-45°C, and the dialysis time is 1-5 days; the molecular weight cutoff of the dialysis bag is 20-40 kDa.

9. A gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity, characterized in that: The method is described in any one of claims 1 to 8.

10. Use of the gelatin / antimicrobial peptide covalent conjugate according to claim 9 in the preparation of antibacterial and / or antibacterial products.

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